1. Field of the Invention
This invention relates to an O-methyltransferase gene cloned from sorghum, the sorghum O-methyltransferase-3 gene, SbOMT3; a construct containing the gene and its promoter; a vector containing the gene; sorghum O-methyltransferase-3 protein, SbOMT3; a method of making SbOMT3 protein; a method of transforming plants; and transgenic plants which express SbOMT3 and thereby convert resveratrol into pterostilbene in planta.
2. Description of the Relevant Art
Resveratrol is a grape phytoalexin that protects plants against important plant pathogens such as Botrytis cinerea (Adrian et al. 1997. J. Chem. Ecol. 23: 1689-1702; Hoos and Blaich. 1990. J. Phytopathol. 129: 102-110). This metabolite is also beneficial to human health by acting as a strong antioxidant (Martinez and Moreno. 2000. Biochem. Pharmacol. 59: 865-870; Stivala et al. 2001. J. Biol. Chem. 276: 22586-22594) and by having cancer chemopreventive properties (Jang et al. 1997. Science 275: 218-220). Pterostilbene, a methylated analogue of resveratrol was demonstrated to have similar antioxidant and cancer chemopreventive properties as resveratrol (Rimando et al. 2002. J. Agric. Food Chem. 50: 3453-3457). Pterostilbene has been shown to have beneficial pharmaceutical and nutraceutical effects for humans and animals. It has been reported to be cytotoxic against a number of cancer cell lines including human breast cancer and murine lymphoid neoplasma cell lines (Rimando et al. 1994. Nat. Prod. Lett. 4: 267-272), and to lower blood glucose level in streptozidin-induced diabetic mice (Manickam et al. 1997. J. Nat. Prod. 60: 609-610). Pterostilbene is more fungitoxic than resveratrol against the plant pathogens Botrytis cinerea, Cladosporium cucumerinum, Pellicularia sasakii, Piricularia oryzae, and Plasmopora viticola (Langcake et al. 1979. Phytochemistry 18: 1025-1027). Pterostilbene also inhibited fungi involved in the chronic disease of grapevines, esca, (Phaeoacremonium aleophilum, Phaeomoniella chlamydospora, Libertella blepharis, Fomitiporia punctata and Stereum hirsutum), whereas resveratrol did not inhibit mycelial growth of these fungi (Mazzullo et al. 2000. Phytopathol. Mediterr. 39: 357-365). Furthermore, pterostilbene was inhibitory against the human pathogens Candida albicans, Cryptococcus neoformans, Staphylococcus aureus, and methicillin-resistant S. aureus, while resveratrol was ineffective against those disease agents (Rimando et al. 2003a. Abstracts of Papers: 219th American Chemical Society National Meeting: AGFD-081).
Sorghum bicolor (L.) Moench is one of the most important cereal crops worldwide (Doggett, H.1988. Sorghum, 2nd Edition, Blackwell Publishing, Ames, Iowa), surpassed only by wheat, rice, corn and barley in total acreage, with the United States currently accounting for a major portion of total world production and exports (FAOSTAT data, faostat.fao.org; accessed March 2006). Allelopathy, the chemical inhibition of one plant species by another, represents a form of chemical warfare between neighboring plants competing for limited light, water, and nutrient resources (Inderjit and Duke. 2003. Planta 217: 529-539; Bais et al. 2004. Trends Plant Sci. 9: 26-32). The allelopathic properties of sorghum were first suggested from observations of reduced growth of other crop species when grown in rotation; moreover certain sorghum species such as Sudan grass (Sorghum sudanese) can produce largely weed-free monocultures without the use of synthetic herbicides (reviewed by Duke et al. 2005. Outlooks Pest Management 16: 64-68). Allelopathic interactions have been proposed to have profound effects on the evolution of plant communities through the loss of susceptible species via chemical interference, and by imposing selective pressure favoring individuals resistant to inhibition from a given allelochemical (e.g., Schulz and Wieland. 1999. Chemoecology 9: 133-141; Bais et al., supra). In addition, allelochemicals released by grain crop species such as barley, rye, and sorghum are thought to play a significant role in their efficacy as weed suppressants when used as cover crops or within intercropping systems (Duke et al. 2002. J. Pestic. Sci. 27: 298-306; Weston and Duke. 2003. Crit. Rev. Plant Sci. 22: 367-389).
Current evidence suggests that a family of allelochemicals active at micromolar concentrations, referred to as sorgoleones, may account for much of the allelopathic properties of Sorghum spp. (Netzly and Butler. 1986. Crop Sci. 26: 775-780; Einhellig and Souza. 1992. J. Chem. Ecol. 18: 1-11; Czarnota et al. 2001. Weed Technol. 15: 813-825). The term sorgoleone is most frequently used to describe the compound corresponding to the predominant congener identified in sorghum root exudates (Kagan et al. 2003. J. Agric. Food Chem. 51: 7589-7595), 2-hydroxy-5-methoxy-3-[(Z,Z)-8′,11′,14′-pentadecatriene]-p-benzoquinone (FIG. 1), which has been estimated to account for as much as 85% of the exudate material (w/w) in some varieties (Czarnota et al. 2001, supra). The remaining exudate largely consists of sorgoleone congeners differing in the length or degree of saturation of the aliphatic side chain, and in the substitution pattern of the quinone ring (Kagan et al., supra; Rimando et al. 2003. J. Nat. Prod. 66: 42-45). The fact that sorgoleone acts as a potent broad-spectrum inhibitor active against many agronomically important monocot and dicot weed species, exhibits a long half-life in soil, and appears to effect multiple targets in vivo (e.g., Netzly and Butler. 1986, supra; Einhellig and Souza, supra; Nimbal et al. 1996. J. Agric. Food Chem. 44: 1343-1347; Rimando et al. 1998. J. Nat. Prod. 61: 927-930; Czarnota et al. 2001, supra; Bertin et al. 2003 Plant Soil 256: 67-83; Duke, S. O. 2003. Trends Biotechnol. 21: 192-195) may make it promising for development as a natural product alternative to synthetic herbicides (Duke, supra).
Both pterostilbene and sorgoleone have important roles in food crops; namely, pterostilbene has been shown to have beneficial pharmaceutical and nutraceutical effects for humans and animals and the allelopathic interactions of sorgoleone can suppress the growth and effects of weeds. Thus, knowing the genetic determinants of pterostilbene biosynthesis and sorgoleone biosynthesis could provide a novel and powerful tool, for enrichment of pterostilbenes and sorgoleones in important food crops. Increasing pterostilbene content in staple crops such blueberries, cranberries, and grapes could help in the alleviation of high cholesterol levels in patients, a condition which affects a large percentage of the world's population. Furthermore, the SbOMT3 gene could be used to alter food plants to selectively modify pterostilbene content and/or composition to provide protection against certain other diseases such as cancer. Thus, the sorghum O-methyltransferase can be used to generate transgenic plants with enhanced levels of pterostilbene in species such as grapes and blueberries that produce resveratrol (Adrian et al. 2000. J. Agric. Food Chem. 48: 6103-6105; Pezet and Pont. 1988. Plant Physiol. Biochem. (Paris) 26: 603-607; Rimando et al. 2000. Abstracts of Papers: 220th American Chemical Society National Meeting: AGFD-074; Rimando and Barney. 2003b. Abstracts of Papers: The 3rd World Congress on Medicinal and Atomatic Plants for Human Welfare: PP10-07) resulting in more disease resistant varieties that also have higher nutraceutical value. It is a primary object of this invention to provide a molecular tool and method for increasing accumulation of pterostilbenes in plant cells.
In addition to utilizing the SbOMT3 gene to generate increased levels of pterostilbenes in plants which already produce resveratrol, i.e., plants such as blueberries, grapes and cranberries, the SbOMT3 gene can be used in combination with a resveratrol synthase gene to produce pterostilbene in any plant species. The enzymatic production of resveratrol in plants requires only the presence of the ubiquitous substrates p-coumaryl-CoA and malonyl-CoA (Austin and Noel. 2003. Nat. Prod. Rep. 20: 79-110); therefore, a resveratrol synthase gene such as the stilbene synthase gene from Arachis hypogaea (peanut; Lanz et al. 1991. J. Biol. Chem. 15: 9971-9976) can be used to generate pterostilbene in planta. The resulting transgenic plants expressing both transgenes, i.e., O-methyltransferase (SbOMT3) and stilbene synthase, simultaneously, would exhibit increased resistance to pathogen infection and also produce fruit that has higher nutraceutical value. This strategy could also be deployed in species where resveratrol is in limiting quantities, to achieve higher accumulation levels of pterostilbene.
We have isolated SbOMT3, a novel O-methyltransferase gene from Sorghum bicolor. The recombinant enzyme, SbOMT3, has been shown in vitro and in vivo to use resveratrol as a substrate to produce pterostilbene.